EP2873730A1

EP2873730A1 - Biological method for producing cis-5-hydroxy-l-pipecolinic acid

Info

Publication number: EP2873730A1
Application number: EP13804143.9A
Authority: EP
Inventors: Tadashi Fujii; Keisuke Tamura
Original assignee: Microbiopharm Japan Co Ltd
Current assignee: Microbiopharm Japan Co Ltd
Priority date: 2012-06-13
Filing date: 2013-06-12
Publication date: 2015-05-20
Also published as: CA2876558C; JP6276178B2; CN111485008A; CN104395466A; US9611490B2; US20150211035A1; EP2873730A4; WO2013187438A1; JPWO2013187438A1; CA2876558A1

Abstract

The present invention provides a method for producing cis-5-hydroxy-L-pipecolic acid. In the production method of the present invention, a gene recombinant microorganism enabling direct production of cis-5-hydroxy-L-pipecolic acid can be used. The present invention also provides such a gene recombinant microorganism. A preferred embodiment of the present invention is characterized in that a gene recombinant microorganism having DNAs encoding proteins involved in the biosynthesis of L-pipecolic acid and a DNA encoding a protein having the L-pipecolic acid cis-5-hydroxylase activity is cultured in a medium, and cis-5-hydroxy-L-pipecolic acid is collected from the medium.

Description

Technical Field

The present invention relates to a gene recombinant microorganism having an ability to produce cis-5-hydroxy-L-pipecolic acid, and a method for producing cis-5-hydroxy-L-pipecolic acid using the microorganism.

Background Art

Cis-5-hydroxy-L-pipecolic acid is a kind of modified amino acid, which has a structure consisting of L-pipecolic acid introduced with hydroxyl group, and it is a substance useful as a synthetic intermediate material of drugs.
Biological methods for producing L-pipecolic acid (or 2-piperidinecarboxylic acid or L-homoproline) have already been reported (Non-patent documents 1, 2, and Patent document 1). In these reports, L-pipecolic acid is produced from L-lysine by using Escherichia coli having the following polynucleotides (also referred to as DNAs).

a) A polynucleotide encoding a protein having the L-lysine 6-aminotransferase enzyme activity
b) A polynucleotide encoding a protein having the pyrroline-5-carboxylate reductase enzyme activity

In these reports, the lat gene (SEQ ID NO: 1) derived from Flavobacterium lutescens IFO3084 strain is mentioned as an example of the polynucleotide of a) mentioned above, and the proC gene (SEQ ID NO: 3) derived from Escherichia coli is mentioned as an example of the polynucleotide of b) mentioned above. Since Escherichia coli originally has the proC gene, an Escherichia coli strain introduced with the lat gene and able to express it has an L-pipecolic acid-producing ability. Further, there have also been reported that the production rate of L-pipecolic acid was improved in Escherichia coli also having a DNA encoding a protein having the lysine-specific permease activity, for example, the lysP gene derived from Escherichia coli (SEQ ID NO: 4).
It has been reported that the CAC47686 protein derived from the alfalfa leguminous bacterium, Sinorhizobium meliloti 1021, has an ability to convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid (Non-patent document 3). The amino acid sequence of this protein is registered at the database GenBank with an accession number of CAC47686. The nucleotide sequence encoding it is registered at the database GenBank with an accession number of AL591792 (SEQ ID NO: 6).
It has also been reported that the BAB52605 protein or CAC47686 protein derived from the Lotus corniculatus var. japonicus leguminous bacterium, Mesorhizobium loti MAFF303099, has an ability to convert L-proline into cis-4-hydroxyproline (Patent document 2). The amino acid sequence of the BAB52605 protein is registered at the database GenBank with an accession number of BAB52605. The nucleotide sequence encoding it is registered at the database GenBank with an accession number of BA000012 (SEQ ID NO: 7, loti gene).

Prior art references

Patent documents

Patent document 1: WO2001/048216 (Japanese Patent No. 4516712 )
Patent document 2: WO2009/139365

Non-patent documents

Non-patent document 1: Biosci. Biotechnol. Biochem., 66 (3), 622-627, 2002
Non-patent document 2: Biosci. Biotechnol. Biochem., 66 (9), 1981-1984, 2002
Non-patent document 3: Adv. Synth. Catal., 353, 1375-1383, 2011

Summary of the Invention

Object to be Achieved by the Invention

It is considered that the CAC47686 protein is a useful enzyme for synthesis of non-natural amino acids, but it suffers from the following problems.
Problem 1) When this protein is expressed in Escherichia coli by using a generally used method, it is insolubilized and inactivated.
Problem 2) When this protein is used for an in vitro reaction, it is promptly denatured.
Problem 3) When this protein is used for an in vitro reaction, in addition to cis-5-hydroxy-L-pipecolic acid, cis-3-hydroxypipecolic acid is also accumulated in an amount substantially equivalent to that of cis-5-hydroxy-L-pipecolic acid, from L-pipecolic acid.
According to Non-patent document 3, in order to express the CAC47686 protein in Escherichia coli, while avoiding the problem 1, for example, a cold shock promoter is used to induce expression of the protein at a low temperature, and GroEL/GroES of Streptomyces coelicolor is coexpressed. Further, it also mentions an idea that hydroxylation of L-pipecolic acid is attained by exposing L-pipecolic acid to live cells of the Escherichia coli expressing the foregoing protein as one of the methods for avoiding the problem 2. However, it does not describe whether this method was actually effective. Further, any method for avoiding the problem 3 has not been proposed. On the basis of the findings described above, it was considered that production of cis-5-hydroxy-L-pipecolic acid using Escherichia coli expressing the CAC47686 protein was difficult.
It was also considered that, like the CAC47686 protein, the BAB52605 protein also might have an ability to convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid. However, as shown in the examples mentioned in this specification, it was found that the cis-5-hydroxy-L-pipecolic acid productivity of Escherichia coli expressing the BAB52605 protein encoded by the loti gene was comparatively low. The identity of the amino acid sequences of the BAB52605 protein and the CAC47686 protein is 66%.

Means for Achieving the Object

The amino acid sequence of the EFV12517 protein has been registered as a protein derived from Segniliparus rugosus ATCC BAA-974 at the database GenBank with an accession number of EFV12517. The nucleotide sequence encoding it is registered at the database GenBank with an accession number of ACZI01000186 (REGION: 1378 .. 2229) (SEQ ID NO: 8, shortcis gene). The EFV12517 protein is annotated as aspartyl/asparaginyl beta-hydroxylase in the GenBank, and as shown in the examples mentioned in this specification, ability to convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid could not be detected in Escherichia coli expressing the EFV12517 protein. However, it was found that Escherichia coli expressing the protein encoded by the polynucleotide (SEQ ID NO: 2, cis gene) expressed from a position upstream by 48 nucleotides (corresponding to 16 amino acids) from the annotation of the EFV12517 protein had the L-pipecolic acid cis-5-hydroxylase activity, and thus could convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid, and the present invention was accomplished.
The present invention provides the followings.

[1] A method for producing cis-5-hydroxy-L-pipecolic acid or a pharmacologically acceptable salt thereof, or a solvate thereof, which comprises the step of producing cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate with a microorganism containing any one of the polynucleotides of the following (A) to (F) in an expressible state:
1. (A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2,
2. (B) a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and encoding a protein having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
3. (C) a polynucleotide showing an identity of 85% or higher to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
4. (D) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25;
5. (E) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
6. (F) a polynucleotide encoding a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.
[2] The production method according to [1], wherein the microorganism further contains a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate, each in an expressible state; and
which further comprises the step of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and then converting L-aminoadipate-delta-semialdehyde into delta-1-piperideine-6-carboxylic acid; and
the step of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate.
[3] The production method according to [2], wherein the polynucleotide encoding a protein having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate is derived from Flavobacterium lutescens.
[4] The production method according to [2] or [3], wherein the microorganism is Escherichia coli, and originally has a polynucleotide encoding a protein having the activity of catalyzing the reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate.
[5] A polynucleotide selected from the polynucleotides of the following (A) to (F):
1. (A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2,
2. (B) a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and encoding a protein having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
3. (C) a polynucleotide showing an identity of 85% or higher to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
4. (D) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25;
5. (E) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
6. (F) a polynucleotide encoding a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.
[6] A vector for transformation of a microorganism, which contains the polynucleotide according to [5].
[7] The vector for transformation of a microorganism according to [6], which further contains a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate.
[8] The vector for transformation of a microorganism according to [6] or [7], which further contains a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating α-ketoglutaric acid, and/or a protein having an activity of catalyzing a reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate.
[9] A gene recombinant microorganism transformed with the vector according to any one of [6] to [8].
[10] A gene recombinant Escherichia coli, which is transformed with the polynucleotide according to [5], and a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and has an ability to generate cis-5-hydroxy-L-pipecolic acid by using L-lysine as a starting material.
[11] A protein selected from the proteins of the following (d) to (f):
- (d) a protein having the amino acid sequence of SEQ ID NO: 25;
- (e) a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
- (f) a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.
[12] A method for producing cis-5-hydroxy-L-pipecolic acid or a pharmacologically acceptable salt thereof, or a solvate thereof, which comprises the step of allowing the protein according to [11] to act on L-pipecolic acid to generate cis-5-hydroxy-L-pipecolic acid.
[13] The production method according to [12], which further comprises:
- the step of allowing a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate to act on L-lysine to generate L-aminoadipate-delta-semialdehyde, and then converting L-aminoadipate-delta-semialdehyde into delta-1-piperideine-6-carboxylic acid; and
- the step of allowing a protein having an activity of catalyzing a reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate to act on the obtained delta-1-piperideine-6-carboxylic acid to generate L-pipecolic acid.
[14] The gene recombinant microorganism or gene recombinant Escherichia coli according to [9] or [10], which has an ability to produce 50 mg or more of cis-5-hydroxy-L-pipecolic acid per 1 L of culture medium.

Brief Description of the Drawings

[Fig. 1] Fig. 1 shows an example of the production method of the present invention, indicating the conversion pathway from L-lysine to cis-5-hydroxy-L-pipecolic acid.
[Fig. 2] Fig. 2 shows the plasmid pRSF-Cis (refer to Example 1).
[Fig. 3] Fig. 3 shows the HPLC charts of the products of the BL21(DE3)/pRSF-Cis strain (refer to Example 2).
[Fig. 4] Fig. 4 shows the LC/MS charts of the products of the BL21(DE3)/pRSF-Cis strain (refer to Example 2). The upper three charts are for 200 µg/mL of authentic samples, and the lower two charts are for the products of the BL21(DE3)/pRSF-Cis strain.
[Fig. 5] Fig. 5 shows the nucleotide sequences and amino acid sequences of the enzyme Lat, enzyme Cis, and mutant Cis.

Modes for Carrying out the Invention

The present invention provides a method for producing cis-5-hydroxy-L-pipecolic acid represented by the following structural formula (I) or a pharmacologically acceptable salt thereof, or a solvate thereof.
The method for producing cis-5-hydroxy-L-pipecolic acid of the present invention can comprise the following steps (1) to (3):

(1) the step of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and then converting L-aminoadipate-delta-semialdehyde into delta-1-piperideine-6-carboxylic acid;
(2) the step of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate; and
(3) the step of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.

The method for producing cis-5-hydroxy-L-pipecolic acid of the present invention comprises the step (3) mentioned above. The step (3) is a step of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate with a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate, namely, the L-pipecolic acid cis-5-hydroxylase (Cis). This step can be biologically carried out by making an organism having the cis gene express Cis.
In the present invention, as the cis gene or the protein, any one of the polynucleotides of the following (A) to (F), or any one of the proteins of the following (d) to (f) can be used:

(A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2,
(B) a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
(C) a polynucleotide showing an identity of 85% or higher to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
(D) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25;
(E) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
(F) a polynucleotide encoding a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
- (d) a protein having the amino acid sequence of SEQ ID NO: 25;
- (e) a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;
- (f) a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.

The nucleotide sequence of cis and the encoded amino acid sequence, which were identified by the inventors of the present invention and used in the examples mentioned in this specification, are shown as SEQ ID NOS: 2 and 25 in Sequence Listing.
Sequences showing high identity to the full length amino acid sequence of the Cis protein shown as SEQ ID NO: 25 were searched for, and it was found that the amino acid sequence shows an identity of 34% to the amino acid sequence of the CAC47686 protein encoded by the nucleotide sequence of SEQ ID NO: 6 (also referred to as "Meliloti protein" in the present invention), and an identity of 33% to the amino acid sequence of the BAB52605 protein encoded by the nucleotide sequence of SEQ ID NO: 7 (also referred to as "Loti protein" in the present invention). More precisely, the results of the identity analysis were Score = 163 bits (413), Expect = 6e-45, Method: Compositional matrix adjust, Identities = 93/275 (34%), Positives = 146/275 (53%), and Gaps = 9/275 (3%) for the former, and Score = 159 bits (402), Expect = 3e-43, Method: Compositional matrix adjust, Identities = 87/260 (33%), Positives = 139/260 (53%), and Gaps = 6/260 (2%) for the latter. Any other sequences showing identity higher than these were not retrieved. For the confirmation of the identity, blastp provided by NCBI was used.
The Cis protein used in the examples comprises an upstream region of the EFV12517 protein (also referred to as "Shortcis protein" in the present invention, which is encoded by the nucleotide sequence of SEQ ID NO: 8, and annotated as aspartyl/asparaginyl beta-hydroxylase at GenBank) encoded by 48 nucleotides, i.e., consisting of 16 amino acid residues. However, the ability to convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid was not known for the Shortcis protein itself, and in fact, it could not be detected for it (refer to Example 2).
Further, the Meliloti protein, which shows an amino acid sequence identity of 34% to the Cis protein used in the examples, is known to have the ability to convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid (Non-patent document 3 mentioned above), but it has been found that it has various problems as described above. On the other hand, according to the studies of the inventors of the present invention, it has been found that the Loti protein, which shows an amino acid sequence identity of 33% to the Cis protein used in the examples, shows comparatively low cis-5-hydroxy-L-pipecolic acid productivity, when it is expressed in Escherichia coli (refer to Example 3). In addition, the amino acid sequence identity between the Loti (BAB52605) protein and the Meliloti (CAC47686) protein is 66%, and thus the same difficulties as those found for the Meliloti protein are expected for the expression of the Loti protein in Escherichia coli, and the production of cis-5-hydroxy-L-pipecolic acid using the obtained protein.
According to one embodiment of the present invention, the cis gene and the encoded protein may be derived from a bacterium of the genus Segniliparus, more specifically, they may be derived from Segniliparus rugosus, and still more specifically, they may be derived from Segniliparus rugosus ATCC BAA-974.
The polynucleotides of (B) to (F) and proteins of (e) to (f) mentioned above should be called mutants of the cis gene and Cis protein used in the examples. Those skilled in the art can appropriately design such mutants in consideration of information on motif of the Cis protein used in the examples etc., and the fact that lack of the upstream sequence of 16 amino acid residues results in lack of the desired activity. According to the studies of the inventors of the present invention, it was found that the Cis protein used in the examples has the aspartyl/asparaginyl beta-hydroxylase region (positions 26 to 174), and L-proline 3-hydroxylase C-terminal region (positions 190 to 274). If a sequence is known, motif analysis thereof can be easily carried out by those skilled in the art by utilizing a website opened to public, for example, Pfam of GenomeNet (http://www.genome.jp/), or the like. Further, whether a certain protein has the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate can be appropriately evaluated by those skilled in the art with referring to the descriptions of the present specification.
According to one embodiment of the present invention, a mutant Cis is used. An example of such a mutant Cis is the protein used in the examples of this specification, which consists of the amino acid sequence of SEQ ID NO: 26 encoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 23. The nucleotide sequence of SEQ ID NO: 23 differs from the nucleotide sequence of cis (SEQ ID NO: 2) used in the examples in two nucleotides (among 897 nucleotides), and the amino acid sequence of SEQ ID NO: 26 differs from the amino acid sequence of Cis (SEQ ID NO: 25) used in the examples in one amino acid (among 299 amino acids). The identity between the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 25 is 99.7%.
The amino acid sequence (SEQ ID NO: 26) of the mutant Cis used in the examples shows an identity of 34% to the amino acid sequence of the Meliloti protein encoded by the nucleotide sequence of SEQ ID NO: 6, and an identity of 29% to the amino acid sequence of the Loti protein encoded by the nucleotide sequence of SEQ ID NO: 7.
Concerning the expression "hybridizable under stringent conditions" used for a polynucleotide in the present invention, for any polynucleotide, the conditions of hybridization can be appropriately selected depending on the polynucleotide to be obtained, according to the descriptions of Molecular Cloning A Laboratory Manual 2nd ed. (Sambrook et al., Cold Spring Harbor Laboratory Press), and Hybridization of Nucleic Acid Immobilization on Solid Supports (ANALYTICAL BIOCHEMISTRY 138, 267-284 (1984)), unless especially indicated. For example, when a DNA showing an identity of 85% or higher is to be obtained, hybridization can be performed at 45°C in the presence of an SSC solution of 2-fold concentration and 50% formamide, and then the filter can be washed at 60°C with an SSC solution of 0.1-fold concentration (SSC solution of 1-fold concentration has a composition of 150 mM sodium chloride and 15 mM sodium citrate). Further, when a DNA showing an identity of 95% or higher is to be obtained, hybridization can be performed at 50°C in the presence of an SSC solution of 2-fold concentration and 50% formamide, and then the filter can be washed at 65°C with an SSC solution of 0.1-fold concentration.
In the present invention, when the expression "amino acid sequence including substitution, deletion, insertion and/or addition of one or more amino acid residues" is used for a protein, for any proteins, the number of the amino acid residues to be substituted, or the like is not particularly limited so long as the protein consisting of the amino acid sequence has the desired function, unless especially indicated. However, the number may be about 1 to 9, or about 1 to 4, or in the case of substitution or the like of amino acid residues having similar properties, substitution or the like may occur for a further larger number of amino acid residues. Means for preparing a polynucleotide for a protein having such an amino acid sequence as mentioned above or the protein itself are well known to those skilled in the art.
When the term "identity" is used for nucleotide sequences or amino acid sequences in the present invention, for any nucleotide sequence or amino acid sequence, it means percentage of the number of the same nucleotide or amino acid residues commonly found at the corresponding positions in the two sequences optimally aligned, unless especially indicated. That is, the identity can be calculated in accordance with the following equation: Identity = (Number of positions at which the same residues locate in two sequences/Total number of positions) x 100, and can be calculated by using a marketed algorithm. Such an algorithm is incorporated into the programs NBLAST and XBLAST described in Altschul et al., J. Mol. Biol., 215 (1990) 403-410. More precisely, search and analysis concerning identity of nucleotide sequence or amino acid sequence can be performed by using an algorithm or program well known to those skilled in the art (for example, BLASTN, BLASTP, BLASTX, ClustalW). The parameters used for executing each program can be appropriately chosen by those skilled in the art, or default parameters of each program may also be used. Specific procedures of these analysis methods are also well known to those skilled in the art.
In this specification, the term identity used for nucleotide sequences or amino acid sequences means, in any case, a sequence identity of at least 70%, preferably 80% or higher, more preferably 85% or higher, still more preferably 90% or higher, further preferably 95% or higher, still further preferably 97.5% or higher, particularly preferably 99% or higher.
The polynucleotides or genes, and proteins or enzymes used in the present invention can be prepared by those skilled in the art by using conventional techniques.
The method for producing cis-5-hydroxy-L-pipecolic acid of the present invention may comprise the step (1) mentioned above. The step (1) is a step of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate by using a protein having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, namely, L-lysine 6-aminotransferase (Lat); and then converting L-aminoadipate-delta-semialdehyde into delta-1-piperideine-6-carboxylic acid. This step can be biologically performed by making an organism having the lat gene express Lat.
In the present invention, as the lat gene or the encoded protein, any one of the polynucleotides of the following (A') to (F'), or any one of the proteins of the following (d') to (f) can be used.

(A') a polynucleotide having the nucleotide sequence of SEQ ID NO: 1,
(B') a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and encoding a protein having an activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate;
(C') a polynucleotide showing an identity of 85% or higher to the nucleotide sequence of SEQ ID NO: 1, and encoding a protein having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate;
(D') a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 24;
(E') a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 24 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate; (F') a polynucleotide encoding a protein comprising an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 24, and having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate;
- (d') a protein having the amino acid sequence of SEQ ID NO: 24;
- (e') a protein having the amino acid sequence of SEQ ID NO: 24 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate;
- (f) a protein having an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 24, and having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate.

The nucleotide sequence of lat and the encoded amino acid sequence derived from Flavobacterium lutescens IFO3084 are shown as SEQ ID NOS: 1 and 22 in Sequence Listing, respectively.
In the present invention, as the lat gene or the encoded protein, those derived from various organisms can be used. According to one embodiment of the present invention, the lat gene and the protein may be derived from a Flavobacterium bacterium, more specifically, Flavobacterium lutescens, further specifically, Flavobacterium lutescens IFO3084.
The method for producing cis-5-hydroxy-L-pipecolic acid of the present invention may comprise the step (2) mentioned above. The step (2) is a step of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate using a protein having the activity of catalyzing the reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate, namely, pyrroline-5-carboxylate reductase (ProC). This step can be biologically carried out by using an organism having the proC gene.
The nucleotide sequence of proC and the encoded amino acid sequence derived from Escherichia coli are shown as SEQ ID NOS: 3 and 25 in Sequence Listing, respectively. As for proC, Patent document 1 mentioned above can be referred to.
ProC is an enzyme originally possessed by Escherichia coli. When the production method of the present invention is implemented by using Escherichia coli, as the enzyme used in the step (2), ProC originally possessed by Escherichia coli may be used, or for the purpose of enhancement of the enzyme or the like, a foreign ProC may be used. When the expression "contained in an expressible state" is used for a polynucleotide in the present invention, the polynucleotide is not limited to a foreign polynucleotide, but also may be a polynucleotide originally possessed by the host, unless especially indicated.
Any one or more or all of the aforementioned steps (1) to (3) of the method for producing cis-5-hydroxy-L-pipecolic acid of the present invention can be biologically performed. A typical example of the method biologically performed is the method performed in a microbial cell containing the required genes in expressible states. The microorganism used for the biological method for producing cis-5-hydroxy-L-pipecolic acid can be obtained by transforming a host microorganism with an appropriately constituted vector. The present invention also provides such gene recombinant microorganism and vector. The organism used for implementing the present invention is, for example, a microorganism, more specifically, for example, a procaryote, still more specifically, for example, Escherichia coli.
The term "gene recombinant microorganism" used in the present invention means a microorganism (bacterium, fungus, yeast, filamentous fungus, etc.) obtained by introducing, into a specific microorganism, a gene derived from another organism using a genetic recombination technique, unless especially indicated. The method for introducing a gene used therefor is not limited to a genetic recombination technique using a vector such as plasmid, but also may be such a method as homologous recombination.
According to the present invention, there can be obtained a gene recombinant microorganism having genes encoding a protein having the L-lysine 6-aminotransferase enzyme activity (Lat), a protein having the pyrroline-5-carboxylate reductase enzyme activity (ProC), and a protein having the L-pipecolic acid cis-5-hydroxylase activity (Cis), respectively, in expressible states, and able to directly produce cis-5-hydroxy-L-pipecolic acid from L-lysine. Further, by culturing such a gene recombinant microorganism and collecting cis-5-hydroxy-L-pipecolic acid from the culture medium, cis-5-hydroxy-L-pipecolic acid can be efficiently produced.
In any embodiment, the gene recombinant microorganism provided by the present invention may have a gene encoding a protein having lysine-specific permease protein activity (LysP) in an expressible state. It has been reported that use of LysP improved production rate of L-pipecolic acid in biological methods for producing L-pipecolic acid, and LysP may also be useful in a biological method for producing cis-5-hydroxy-L-pipecolic acid from the same point of view. The nucleotide sequence of the lysP gene derived from Escherichia coli is shown as SEQ ID NO: 4.
In any embodiment, the gene recombinant microorganism provided by the present invention may further contain a gene encoding a protein having an activity of catalyzing a reaction of generating α-ketoglutaric acid in an expressible state. Amino acid hydroxylases, of which typical example is L-pipecolic acid cis-5-hydroxylase, require α-ketoglutaric acid for the hydroxylation reaction catalyzed thereby (Non-patent document 3). Further, it is also known that L-lysine 6-aminotransferase also requires α-ketoglutaric acid for the transamination reaction catalyzed thereby, and converts it into glutamic acid (EC 2.6.1.36). Therefore, when L-pipecolic acid is produced from L-lysine using a microorganism that can express these proteins, it is expected to be important to regenerate α-ketoglutaric acid.
As enzyme that regenerates α-ketoglutaric acid from glutamic acid, glutamate dehydrogenase (EC 1.4.1.2) is known, and the reaction catalyzed by this enzyme can couple with the reaction catalyzed by L-lysine 6-aminotransferase. The nucleotide sequence of the rocG gene derived from Bacillus subtilis subsp. subtilis str. 168 is shown as SEQ ID NO: 5.
Hereafter, embodiments of the present invention will be explained more specifically.
The polynucleotide encoding a protein having the L-pipecolic acid cis-5-hydroxylase activity used for the biological method for producing cis-5-hydroxy-L-pipecolic acid of the present invention can be obtained from a cell of an appropriate microorganism by a method well known in this field (for example, the colony hybridization method described in Molecular Cloning A Laboratory Manual, 2nd ed.). Preferred examples of such a microorganism include a strain belonging to the genus Segniliparus, more specifically a strain belonging to Segniliparus rugosus, still more specifically Segniliparus rugosus ATCC BAA-974. Alternatively, as described in the examples of the present specification, a DNA encoding a protein having the L-pipecolic acid cis-5-hydroxylase activity may be artificially synthesized.
The polynucleotide encoding a protein having the L-pipecolic acid cis-5-hydroxylase activity, which was artificially synthesized in the examples, and expressed from the position upstream from the annotation of the EFV12517 protein by 48 nucleotides (corresponding to 16 amino acid residues) is as shown in SEQ ID NO: 2 mentioned in Sequence Listing. This DNA of SEQ ID NO: 2 contains the open reading frame (ORF) of cis (nucleotides 1 to 897).
The recombinant as one embodiment of the present invention is a gene recombinant microorganism containing polynucleotides encoding enzymes involved in the biosynthesis of L-pipecolic acid (for example, Lat, ProC, LysP, RocG), and a polynucleotide encoding L-pipecolic acid cis-5-hydroxylase (Cis), and it can be produced by incorporating both these DNAs into a host microorganism.
As the host, any microorganism into which the target DNAs can be incorporated, and which can produce the objective cis-5-hydroxy-L-pipecolic acid can be used without any particular restriction. Preferred examples of the microorganism include a strain belonging to Escherichia coli, for example, the Escherichia coli BL21(DE3) strain, and so forth.
The means for incorporating a foreign polynucleotide into a host, and expressing it is not particularly limited, and for example, methods described in Molecular Cloning A Laboratory Manual, 2nd ed., Current Protocols in Molecular Biology (edited by Frederick M. Ausubel et al., 1987), etc. can be used. The host-plasmid vector system is not particularly limited so long as a system that allows stable retention and expression of the objective polynucleotides in a host is chosen. Further, the plasmid may contain, in addition to the target polynucleotides, an autonomously replicable sequence, promoter sequence, terminator sequence, drug resistance gene, and so forth, and as for the type of the plasmid, the plasmid is not limited to autonomously replicable plasmid, but it may be an integration type plasmid having a sequence homologous to a certain region of the genome of the host planned to be used. The objective polynucleotide may be incorporated at any site on the plasmid or genome of the host microorganism.
When Escherichia coli is used as the host, examples of the autonomously replicable vector include pUC19, pRSFDuet-1, and so forth, examples of the promoter sequence include lac, T7, and so forth, examples of the terminator sequence include lacZ terminator, T7 terminator, and so forth, and examples of the drug resistance gene include ampicillin resistance gene, kanamycin resistance gene, and so forth.
When the present invention is implemented with a gene recombinant Escherichia coli, among the proteins involved in the biosynthesis of L-pipecolic acid, introduction of Lat and Cis is important, but whether ProC, LysP, and RocG are introduced or not can be appropriately determined in consideration of amount of the objective product, whether L-pipecolic acid is simultaneously produced or not, and degree thereof, if produced, utilization ratio of L-lysine as the starting material, and so forth.
By culturing gene recombinant microorganisms prepared as described above, evaluating productivities thereof for cis-5-hydroxy-L-pipecolic acid in a conventional manner, and selecting an appropriate recombinant, a useful strain that produces cis-5-hydroxy-L-pipecolic acid can be obtained. The product may be measured by the method described in the examples of the present specification.
The biological method for producing cis-5-hydroxy-L-pipecolic acid of the present invention is typically performed by culturing a gene recombinant microorganism. The culture conditions for the microorganism can be appropriately designed by those skilled in the art depending on the microorganism to be used. When Escherichia coli is used as the host, an appropriate amount of the microorganism can be inoculated into a commonly used medium containing antibiotics as a selection marker as required, and cultured at 20 to 40°C for 6 to 72 hours, preferably 9 to 60 hours, more preferably 12 to 48 hours, with stirring or shaking at 100 to 400 rpm as required to allow proliferation of the cells. By supplying L-lysine or a salt thereof as the starting material, α-ketoglutaric acid or a salt thereof as required, and an appropriate inducer (for example, isopropylthio-β-galactoside (IPTG)) also as required, during or after the culture, and performing culture at 20 to 40°C for 3 to 72 hours, preferably 4 to 60 hours, more preferably 6 to 48 hours, with stirring or shaking at 100 to 400 rpm as required, the objective substance is obtained in the culture medium. Timing of supply of L-lysine etc., and termination point of the culture can be appropriately determined by those skilled in the art in consideration of the production amount of the objective substance etc. For example, L-lysine etc. can be supplied, and culture can be terminated, after lapse of times determined beforehand on the basis of results of culture performed in a smaller scale in advance.
The initial concentration of L-lysine may be, for example, 2 to 32 g/L, more specifically 4 to 16 g/L, and the initial concentration of α-ketoglutaric acid may be, for example, 0 to 16 g/L, more specifically 0 to 8 g/L. Alternatively, the initial concentration of α-ketoglutaric acid may be, for example, 1 to 16 g/L, more specifically 2 to 8 g/L.
A preferred example of the gene recombinant Escherichia coli of the present invention is a gene recombinant Escherichia coli having an ability to produce 50 mg or more of cis-5-hydroxy-L-pipecolic acid per 1 L of culture medium.
Although the present invention may be explained for cis-5-hydroxy-L-pipecolic acid as an example of "cis-5-hydroxy-L-pipecolic acid or a pharmacologically acceptable salt thereof, or a solvate thereof, such explanation is also applied to the pharmacologically acceptable salt of cis-5-hydroxy-L-pipecolic acid and the solvate of them, unless especially indicated, and those skilled in the art can modify the method for producing cis-5-hydroxy-L-pipecolic acid into a method for producing a pharmacologically acceptable salt thereof or a solvate thereof by appropriately adding required steps. The "pharmacologically acceptable salt or a solvate thereof' referred to in the present invention include, as the salt, an alkali metal salt (for example, sodium salt, potassium salt), alkaline earth metal salt (for example, magnesium salt, calcium salt), ammonium salt, mono-, di-, or tri-(lower alkyl or hydroxyalkyl)ammonium salt (for example, ethanol ammonium salt, diethanol ammonium salt, triethanol ammonium salt, tromethamine salt), hydrochloride, hydrobromide, hydroiodide, nitrate, phosphate, sulfate, formate, acetate, citrate, oxalate, fumarate, maleate, succinate, malate, tartrate, trichloroacetate, trifluoroacetate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, mesitylenesulfonate, and naphthalenesulfonate.
Further, the salt may be an anhydride or solvate, and examples of the solvate include hydrate, methanol solvate, ethanol solvate, propanol solvate, and 2-propanol solvate.

Examples

Hereafter, the present invention will be specifically explained with reference to examples. However, the present invention is not limited by these examples.

Example 1: Construction of pRSFduet-Cis

With reference to the nucleotide sequence of SEQ ID NO: 1, a primer lac-lat-NcoF2 (refer to SEQ ID NO: 9) having the NcoI site at the 5' end, and a primer lat-XhoR (refer to SEQ ID NO: 10) having the SpeI site at the 5' end were designed and prepared. Then, PCR was performed by using these two kinds of primers and the genomic DNA of the Flavobacterium lutescens IFO3084 strain as the template. PCR was performed by using KOD-Plus-Ver.2 (TOYOBO) with 30 cycles of two-step reaction comprising denaturation at 98°C for 20 seconds, annealing at 60°C for 20 seconds, and extension at 68°C for 90 seconds. A DNA fragment having a size of about 1.5 kbp and containing lat was collected from the PCR amplification reaction mixture by using Wizard PCR Preps DNA Purification System (Promega). The obtained DNA fragment was digested with the restriction enzymes NcoI and XhoI to obtain a lat fragment.
With reference to the nucleotide sequence of SEQ ID NO: 4, a primer lysP-SD-XhoF (refer to SEQ ID NO: 11) having the XhoI site at the 5' end, and a primer lysP-KpnR (refer to SEQ ID NO: 12) having the KpnI site at the 5' end were designed and prepared (SIGMA GENOSYS). Then, PCR was performed in the same manner as mentioned above by using these two kinds of primers and the genomic DNA of the JM109 strain derived from the Escherichia coli K12 as the template. A DNA fragment having a size of about 1.5 kbp and containing lysP was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes XhoI and KpnI to obtain a lysP fragment.
With reference to the nucleotide sequence of SEQ ID NO: 3, a primer proC-SD-KpnF (refer to SEQ ID NO: 13) having the KpnI site at the 5' end, and a primer proC-BamR (refer to SEQ ID NO: 14) having the BamHI site at the 5' end were designed and prepared. Then, PCR was performed in the same manner as mentioned above by using these two kinds of primers and the genomic DNA of the Escherichia coli K12 JM109 strain as the template. A DNA fragment having a size of about 1.0 kbp and containing proC was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes KpnI and BamHI to obtain a proC fragment.
Four of components, a plasmid digestion product obtained by digesting pRSFDuet-1 (Novergen) with the restriction enzymes NcoI and BamHI, the lat fragment, the lysP fragment, and the proC fragment, were ligated by using DNA Ligation Kit ver.2 (Takara Bio) to construct a plasmid pRSF-LLP having the lat, lysP, and proC genes, and used to transform E. coli JM109 Competent Cells (Takara Bio).
Then, with reference to the nucleotide sequence of SEQ ID NO: 5, a primer rocG-SD-BamF (refer to SEQ ID NO: 15) having the BamHI site at the 5' end, and a primer rocG-XbaR (refer to SEQ ID NO: 16) having the XbaI site at the 5' end were designed and prepared. Then, PCR was performed in the same manner as mentioned above by using these two kinds of primers and the genomic DNA of the Bacillus subtilis subsp. subtilis str. 168 strain as the template. A DNA fragment having a size of about 1.3 kbp and containing rocG was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes BamHI and XbaI to obtain a rocG fragment.
A plasmid digestion product obtained by digesting pRSF-LLP with the restriction enzymes BamHI and XbaI, and the rocG fragment were ligated to construct a plasmid pRSF-PA having the lat, lysP, proC, and rocG genes.
With reference to the nucleotide sequence of SEQ ID NO: 8, a primer segni-short-NdeF (refer to SEQ ID NO: 17) having the NdeI site at the 5' end, and a primer segni-cis-BgIR (refer to SEQ ID NO: 18) having the BglII site at the 5' end were designed and prepared. Then, a gene was artificially synthesized as the nucleotide sequence of SEQ ID NO: 8 (GenScript), and by using this gene as the template, PCR was performed in the same manner as mentioned above. A DNA fragment having a size of about 0.9 kbp and containing cis was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes NdeI and BglII to obtain a cisShort fragment.
A plasmid digestion product obtained by digesting pRSF-PA with the restriction enzymes NdeI and BglII was ligated with the cisShort fragment to construct a plasmid pRSF-CisShort having the lat, lysP, proC, and rocG genes as well as the gene (shortcis) encoding the EFV12517 protein.
With reference to the nucleotide sequence of SEQ ID NO: 2, a primer segni-cis-NdeF2 (refer to SEQ ID NO: 19) having the NdeI site at the 5' end, and a primer segni-cis-BglR (refer to SEQ ID NO: 18) having the BglII site at the 5' end were designed and prepared. Then, a gene was artificially synthesized as the nucleotide sequence of SEQ ID NO: 2 (GenScript), and by using this gene as the template, PCR was performed in the same manner as mentioned above. A DNA fragment having a size of about 0.9 kbp and containing cis was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes NdeI and BglII to obtain a cis fragment.
A plasmid digestion product obtained by digesting pRSF-PA with the restriction enzymes NdeI and BglII was ligated with the cis fragment to construct a plasmid pRSF-Cis having the lat, lysP, proC, rocG, and cis genes (Fig. 2).
With reference to the nucleotide sequence of SEQ ID NO: 1, a primer lac-lat-NcoF2 (refer to SEQ ID NO: 9) having the NcoI site at the 5' end, and a primer lat-(Spe)AflR2 (refer to SEQ ID NO: 20) having the AflII site at the 5' end were designed and prepared. Then, PCR was performed by using these two kinds of primers and the plasmid pRSF-Cis as the template. A DNA fragment having a size of about 1.5 kbp and containing lat was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes NcoI and AflII to obtain a lat2 fragment.
A plasmid digestion product obtained by digesting pRSF-Cis with the restriction enzymes NcoI and AflII was ligated with the lat2 fragment to construct a plasmid pRSF-LatCis having the lat and cis genes.
With reference to the nucleotide sequence of SEQ ID NO: 7, a primer loti-SD-PacF (refer to SEQ ID NO: 21) having the NcoI site at the 5' end, and a primer loti-AvrR (refer to SEQ ID NO: 22) having the AvrII site at the 5' end were designed and prepared. Then, PCR was performed by using these two kinds of primers and the genomic DNA of the Mesorhizobium loti MAFF303099 strain as the template. A DNA fragment having a size of about 0.9 kbp and containing a gene encoding the BAB52605 protein was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes PacI and AflII to obtain a loti fragment.
A plasmid digestion product obtained by digesting pRSF-Cis with the restriction enzymes PacI and AflII was ligated with the loti fragment to construct a plasmid pRSF-Loti having the lat, lysP, proC, rocG genes and a gene (loti) encoding the BAB52605 protein.
Finally, PCR was performed by using segni-cis-NdeF2 and segni-cis-BglR as the primers, as well as pRSF-Cis as the template with Diversify™ PCR Random Mutagenesis Kit (Clonteck) under Condition 5. A DNA fragment having a size of about 0.9 kbp was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzymes NdeI and BglII to obtain a mutant cis fragment. A plasmid digestion product obtained by digesting pRSF-PA with the restriction enzymes NdeI and BglII was ligated with the mutant cis fragment to construct a plasmid pRSF-MutCisLibrary having the lat, lysP, proC, rocG, and mutant cis genes.

Example 2: Cis-5-hydroxy-L-pipecolic acid production test 1

Escherichia coli One Shot BL21(DE3) Competent Cells (Life Technologies Japan) were transformed with each of the plasmids pRSF-Cis (Fig. 2), pRSF-CisShort, pRSF-PA, pRSF-LatCis, and pRSFDuet-1 to obtain BL21(DE3)/pRSF-Cis, BL21(DE3)/pRSF-CisShort, BL21(DE3)/pRSF-PA, BL21(DE3)/pRSF-LatCis, and BL21(DE3)/pRSFDuet-1 strains, respectively. These strains were each inoculated into the M9SEED liquid medium (3.39% Na₂HPO₄, 1.5% KH₂PO₄, 0.25% calcium chloride, 0.5% ammonium chloride, 1% casamino acid, 0.002% thymine, 0.1 mM calcium chloride, 0.1 mM iron sulfate, 0.4% glucose, and 0.001 mM magnesium chloride) containing kanamycin sulfate (25 µg/ml), and cultured at 30°C for 22 hours with shaking at 220 rpm. This culture medium (10 µL) was added to the M9Cis medium (3.39% Na₂HPO₄, 1.5% KH₂PO₄, 0.25% calcium chloride, 0.5% ammonium chloride, 1% casamino acid, 0.002% thymine, 0.1 mM calcium chloride, 0.1 mM iron sulfate, 0.01% 80 µg/ml 5-aminolevulinic acid) containing kanamycin sulfate (30 µg/mL) and Overnight Express Autoinduction Systems (Merck), and culture was performed at 30°C for 9 hours with shaking at 220 rpm. Then, 40% L-lysine hydrochloride (10 µL; final concentration, 8 g/L), 20% α-ketoglutaric acid (10 µL; final concentration, 4 g/L), 100 mM IPTG (0.5 µL; final concentration, 0.1 mM), and 50% glycerol (5 µL; final concentration, 0.5%) were added, and culture was further performed at 30°C with shaking at 220 rpm. Twenty-four hours after the start of the culture, centrifugation supernatant of the culture medium was collected, and used for the preparation of LC/MS analysis samples.
Each sampled solution was FDLA-ized by the following method using Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA, Tokyo Chemical Industry).
To 20 µL of the centrifugation supernatant of the sampled solution diluted 10 time, 1 M NaHCO₃ (6.25 µL) and 1% L-FDLA solution in acetone (30 µL) were added, and the mixture was kept warm at 37°C for 1 hour. The reaction was terminated by adding 1 N HCl (6.25 µL), and acetonitrile (60 µL) was added to the mixture for dilution to obtain an FDLA-ized solution.
Amounts of L-lysine, L-pipecolic acid, and cis-5-hydroxy-L-pipecolic acid contained in the obtained FDLA-ized solution were measured by HPLC and LC/MS. The HPLC and LC/MS analysis charts are shown in Figs. 3 and 4. The quantification results are shown in Table 1. The HPLC and LC/MS measurement conditions are shown below.

Analysis conditions

Column: CAPCELLPAK C18 SG120, 4.6 x 150 mm, 5 µm
Flow rate: 1.0 mL/minute
Mobile phase: A = 0.1% acetic acid, B = acetonitrile Gradient: 0 to 9 minutes (B = 30 to 65%), 9.01 to 12 minutes (B = 90%), 12.01 to 15 minutes (B = 30%)
Detection: 340 nm
Injection volume: 5 µL
Column temperature: 40°C
MS: Agilent 6320 (Ion trap)
Mode: ESI/APCI positive
Scan Range: m/z 100 to 900
Analytical time: 15 minutes
Retention time:
- L-Lysine, 10.0 minutes
- L-Pipecolic acid, 8.5 minutes
- Cis-5-hydroxy-L-pipecolic acid, 5.8 minutes

[Table 1]

Strain	Accumulated amount (g/L)
Strain	Cis-5-hydroxy-L-pipecolic acid	L-Pipecolic acid	L-Lysine
BL21(DE3)/pRSF-Cis	3.4	4.2	0.2
BL21 (DE3)/pRSF-CisShort	N.D.	6.2	0.3
BL21(DE3)/pRSF-LatCis	2.3	0.8	5.6
BL21(DE3)/pRSF-PA	N.D.	6.9	0.2
BL21(DE3)/pRSFDuet-1	N.D.	N.D.	8.9

N.D. means "not detected".

As a result, whereas production of L-pipecolic acid and cis-5-hydroxy-L-pipecolic acid could not be confirmed for the BL21(DE3)/pRSFDuet-1 strain, the BL21(DE3)/pRSF-CisShort strain (containing the lat, lysP, proC, rocG, and shortcis genes on the plasmid) and the BL21(DE3)/pRSF-PA strain (containing the lat, lysP, proC, and rocG genes on the plasmid) produced L-pipecolic acid. Further, the BL21(DE3)/pRSF-Cis strain (containing the lat, lysP, proC, rocG, and cis genes on the plasmid), and the BL21(DE3)/pRSF-LatCis strain (containing the lat and cis genes on the plasmid) produced cis-5-hydroxy-L-pipecolic acid and L-pipecolic acid.
These results demonstrated that introduction of the cis gene into a strain having an L-pipecolic acid-producing ability (coexpression of the lat gene and the cis gene in this case) enables direct production of cis-5-hydroxy-L-pipecolic acid, and further coexpression of the lysP, proC, and rocG genes can improve the productivity. As the standard sample of L-pipecolic acid, L-Pipecolic Acid (Tokyo Chemical Industry) was used, and as the standard sample of cis-5-hydroxy-L-pipecolic acid, (2S,5S)-5-Hydroxypipecolic Acid (SV ChemBIOTECH. INC) was used.
As described above, whereas the ability to convert L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid could not be detected for the Escherichia coli expressing the EFV12517 protein encoded by the shortcis gene, the Escherichia coli expressing the protein encoded by the polynucleotide (cis gene) expressed from the position upstream from the annotation of the EFV12517 protein by 48 nucleotides (corresponding to 16 amino acid residues) had the L-pipecolic acid cis-5-hydroxylase activity for converting L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid. Homologies of the amino acid sequence of the protein encoded by this cis gene to the amino acid sequences of the CAC47686 protein and BAB52605 protein were 34% and 33%, respectively.
On the basis of the aforementioned results, it was considered that it was difficult to expect from known information that Escherichia coli expressing the protein encoded by the cis gene has the L-pipecolic acid cis-5-hydroxylase activity for converting L-pipecolic acid into cis-5-hydroxy-L-pipecolic acid.

Example 3: Cis-5-hydroxy-L-pipecolic acid production test 2

Escherichia coli One Shot BL21(DE3) Competent Cells were transformed with each of the plasmids pRSF-Cis, and pRSF-Loti to obtain strains BL21(DE3)/pRSF-Cis, and BL21(DE3)/pRSF-Loti, respectively. Further, one of the strains obtained by transforming Escherichia coli One Shot BL21(DE3) Competent Cells with the plasmid pRSF-MutCisLibrary was used as BL21(DE3)/pRSF-MutCis1. Culture of these strains and analysis were performed in the same manners as those of Example 2 to measure the amounts of L-pipecolic acid and cis-5-hydroxy-L-pipecolic acid. The measurement results are shown in Table 2.

[Table 2]

Strain	Accumulated amount (g/L)
Strain	Cis-5-hydroxy-L-pipecolic acid	L-Pipecolic acid	L-Lysine
BL21(DE3)/pRSF-Cis	2.9	3.8	N.D.
BL21(DE3)/pRSF-Loti	0.1	6.5	N.D.
BL21(DE3)/pRSF-MutCis1	3.0	3.6	N.D.

N.D. means "not detected".

As a result, the cis-5-hydroxy-L-pipecolic acid production amount of the BL21(DE3)/pRSF-Loti strain (containing the lat, lysP, proC, rocG, and loti genes on the plasmid) was about 1/30 of that of the BL21(DE3)/pRSF-Cis strain (containing the lat, lysP, proC, rocG, and cis genes on the plasmid). This result demonstrated that, when Escherichia coli expressing the BAB52605 protein encoded by the loti gene is used, the amount of cis-5-hydroxy-L-pipecolic acid to be obtained is comparatively small.
On the other hand, even the BL21(DE3)/pRSF-MutCisl strain (containing the lat, lysP, proC, rocG, and mutant cis genes on the plasmid) showed a cis-5-hydroxy-L-pipecolic acid production amount equivalent to or higher than that obtained with the BL21(DE3)/pRSF-Cis strain. The result of the nucleotide sequence analysis of this mutant cis gene performed by using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) is shown as SEQ ID NO: 23. As a result, the homology between the cis gene nucleotide sequence and the mutant cis gene nucleotide sequence was 99.7%, since they were different in 2 nucleotides among the 897 nucleotides in total. This result demonstrated that it is possible to produce cis-5-hydroxy-L-pipecolic acid by using Escherichia coli expressing a protein encoded by a gene showing a homology of 99.7% or higher to the cis gene nucleotide sequence.

Example 4: Cis-5-hydroxy-L-pipecolic acid production test 3

Escherichia coli One Shot BL21(DE3) Competent Cells were transformed with each of the plasmids pRSF-Cis, and pRSF-CisΔproCΔrocG to obtain strains BL21(DE3)/pRSF-Cis, and BL21(DE3)/pRSF-CisΔproCΔrocc-G, respectively. Culture of these strains and analysis were performed in the same manners as those of Example 2 to measure the amounts of L-pipecolic acid and cis-5-hydroxy-L-pipecolic acid. The measurement results are shown in Table 3. [Table 3]

Strain Accumulated amount (g/L)

Cis-5-hydroxy-L-pipecolic acid L-Pipecolic acid L-Lysine

BL21(DE3)/pRSF-Cis 3.8 4.6 0

BL21(DE3)/pRSF-CisΔproCΔrocG 2.6 0 7.6
As a result, the cis-5-hydroxy-L-pipecolic acid production amount of the BL21(DE3)/pRSF-CisΔproCΔrocG strain (containing the lat, lysP, and cis genes on the plasmid) was about 2/3 of that of the BL21 (DE3)/pRSF-Cis strain (containing the lat, lysP, proc, rocG, and cis genes on the plasmid). This result demonstrated that presence of the proC and rocG genes on the plasmid in addition to the lat and lysP genes provides a larger amount of the obtained cis-5-hydroxy-L-pipecolic acid.

Example 5: Cis-5-hydroxy-L-pipecolic acid production test 4

In order to delete the proC gene, rocG gene, or both of these genes of the plasmid pRSF-Cis, the following primers were produced.

Primer proCrocGX-SpeR (refer to SEQ ID NO: 27)
Primer proCX-SpeR (refer to SEQ ID NO: 28)
Primer rocGX-SpeF (refer to SEQ ID NO: 29)
Primer proCX-SpeF (refer to SEQ ID NO: 30)

The plasmid pRSF-CisΔproC corresponding to the plasmid pRSF-Cis of which proC gene is deleted was produced as follows. By using the primer proCX-SpeF and the primer proCrocGX-SpeR, as well as pRSF-Cis as the template, PCR was performed. A DNA fragment corresponding to the plasmid pRSF-Cis of which proC gene is deleted was collected from the PCR amplification reaction mixture. The obtained DNA fragment was digested with the restriction enzyme SpeI, and the product was self-ligated to construct pRSF-CisΔproC, which was used to transform E. coli JM109 Competent Cells (Takara Bio). Similarly, the plasmid pRSF-CisΔrocG corresponding to the plasmid pRSF-Cis of which rocG gene is deleted was prepared by using the primer rocGX-SpeF and the primer proCX-SpeR, and the plasmid pRSF-CisΔproCΔrocG corresponding to the plasmid pRSF-Cis of which proC gene and rocG gene are deleted was produced by using the primer rocGX-SpeF and the primer proCrocGX-SpeR.

Escherichia coli One Shot BL21(DE3) Competent Cells (Life Technologies Japan) were transformed with each of the plasmids pRSF-Cis, pRSF-CisΔproC, pRSF-CisΔrocG, and pRSF-CisΔproCΔrocG to obtain BL21(DE3)/pRSF-Cis, BL21(DE3)/pRSF-CisΔproC, BL21(DE3)/pRSF-CisΔrocG, and BL21(DE3)/pRSF-CisΔproCΔrocG, respectively. These strains were each inoculated into the M9SEED liquid medium (3.39% Na₂HPO₄, 1.5% KH₂PO₄, 0.25% calcium chloride, 0.5% ammonium chloride, 1% casamino acid, 0.002% thymine, 0.1 mM calcium chloride, 0.1 mM iron sulfate, 0.4% glucose, and 0.001 mM magnesium chloride) containing kanamycin sulfate (25 µg/ml), and cultured at 30°C for 9 hours with shaking at 220 rpm. This culture medium (10 µL) was added to the M9Cis medium (3.39% Na₂HPO₄, 1.5% KH₂PO₄, 0.25% calcium chloride, 0.5% ammonium chloride, 1% casamino acid, 0.002% thymine, 0.1 mM calcium chloride, 0.1 mM iron sulfate, 0.01% 80 µg/ml 5-aminolevulinic acid) containing kanamycin sulfate (30 µg/mL), L-lysine hydrochloride (final concentration, 8 g/L), α-ketoglutaric acid (final concentration, 2 g/L) and Overnight Express Autoinduction Systems (Merck), and culture was performed at 30°C for 15 hours with shaking at 220 rpm. Then, 40% L-lysine hydrochloride (5 µL; final concentration, 4 g/L), 20% α-ketoglutaric acid (5 µL; final concentration, 2 g/L), 100 mM IPTG (0.5 µL; final concentration, 0.1 mM), and 50% glycerol (5 µL; final concentration, 0.5%) were added, and culture was further performed at 30°C with shaking at 220 rpm. Thirty-nine hours after the start of the culture, centrifugation supernatant of the culture medium was collected, and used for the preparation of LC/MS analysis samples. The measurement results are shown in Table 4.

[Table 4]

Strain

Accumulated amount (g/L)

	Cis-5-hydroxy-L-pipecolic acid	L-Pipecolic acid	L-Lysine
BL21(DE3)/pRSF-Cis	1.3	1.0	4.2
BL21(DE3)/pRSF-CisΔproC	1.0	1.0	4.4
BL21(DE3)/pRSF-CisΔrocG	1.5	1.2	4.0
BL21(DE3)/pRSF-CisΔproCΔrocG	1.5	1.2	2.7

As a result, under these culture conditions, the cis-5-hydroxy-L-pipecolic acid production amounts obtained with BL21(DE3)/pRSF-CisΔrocG and BL21(DE3)/pRSF-CisΔproCΔrocG were larger than that obtained with BL21(DE3)/pRSF-Cis.

Sequence Listing Free Text

SEQ ID NO: 1, Nucleotide sequence of lat
SEQ ID NO: 2, Nucleotide sequence of cis
SEQ ID NO: 3, Nucleotide sequence of proC
SEQ ID NO: 4, Nucleotide sequence of lysP
SEQ ID NO: 5, Nucleotide sequence of rocG
SEQ ID NO: 6, Nucleotide sequence of meliloti
SEQ ID NO: 7, Nucleotide sequence of loti
SEQ ID NO: 8, Nucleotide sequence of shortcis
SEQ ID NO: 9, Primer lac-lat-NcoF2
SEQ ID NO: 10, Primer lat-XhoR
SEQ ID NO: 11, Primer lysP-SD-XhoF
SEQ ID NO: 12, Primer lysP-KpnR
SEQ ID NO: 13, Primer proC-SD-KpnF
SEQ ID NO: 14, Primer proC-BamR
SEQ ID NO: 15, Primer rocG-SD-BamF
SEQ ID NO: 16, Primer rocG-XbaR
SEQ ID NO: 17, Primer segni-short-NdeF
SEQ ID NO: 18, Primer segni-cis-BglR
SEQ ID NO: 19, Primer segni-cis-NdeF2
SEQ ID NO: 20, Primer lat-(Spe)AflR2
SEQ ID NO: 21, Primer loti-SD-PacF
SEQ ID NO: 22, Primer loti-AvrR
SEQ ID NO: 23, Nucleotide sequence of mutant cis
SEQ ID NO: 24, Amino acid sequence of the protein encoded by lat
SEQ ID NO: 25, Amino acid sequence of the protein encoded by cis
SEQ ID NO: 26, Amino acid sequence of the protein encoded by mutant cis
SEQ ID NO: 27, Primer proCrocGX-SpeR
SEQ ID NO: 28, Primer proCX-SpeR
SEQ ID NO: 29, Primer rocGX-SpeF
SEQ ID NO: 30, Primer proCX-SpeF

Claims

A method for producing cis-5-hydroxy-L-pipecolic acid or a pharmacologically acceptable salt thereof, or a solvate thereof, which comprises the step of producing cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate with a microorganism containing any one of the polynucleotides of the following (A) to (F) in an expressible state:
(A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2,

(B) a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and encoding a protein having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(C) a polynucleotide showing an identity of 85% or higher to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(D) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25;

(E) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(F) a polynucleotide encoding a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.
The production method according to claim 1, wherein the microorganism further contains a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate, each in an expressible state; and
which further comprises the step of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and then converting L-aminoadipate-delta-semialdehyde into delta-1-piperideine-6-carboxylic acid; and
the step of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate.
The production method according to claim 2, wherein the polynucleotide encoding a protein having the activity of catalyzing the reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate is derived from Flavobacterium lutescens.
The production method according to claim 2 or 3, wherein the microorganism is Escherichia coli, and originally has a polynucleotide encoding a protein having the activity of catalyzing the reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate.
A polynucleotide selected from the polynucleotides of the following (A) to (F):
(A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 2,

(B) a polynucleotide hybridizable with a polynucleotide comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions, and encoding a protein having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(C) a polynucleotide showing an identity of 85% or higher to the nucleotide sequence of SEQ ID NO: 2, and encoding a protein having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(D) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25;

(E) a polynucleotide encoding a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(F) a polynucleotide encoding a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.
A vector for transformation of a microorganism, which contains the polynucleotide according to claim 5.
The vector for transformation of a microorganism according to claim 6, which further contains a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate.
The vector for transformation of a microorganism according to claim 6 or 7, which further contains a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating α-ketoglutaric acid, and/or a protein having an activity of catalyzing a reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate.
A gene recombinant microorganism transformed with the vector according to any one of claims 6 to 8.
A gene recombinant Escherichia coli, which is transformed with the polynucleotide according to claim 5, and a polynucleotide encoding a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate, and has an ability to generate cis-5-hydroxy-L-pipecolic acid by using L-lysine as a starting material.
A protein selected from the proteins of the following (d) to (f):
(d) a protein having the amino acid sequence of SEQ ID NO: 25;

(e) a protein having the amino acid sequence of SEQ ID NO: 25 including substitution, deletion, insertion, and/or addition of one or more amino acid residues, and having an activity of catalyzing a reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate;

(f) a protein consisting of an amino acid sequence showing an identity of 85% or higher to the amino acid sequence of SEQ ID NO: 25, and having the activity of catalyzing the reaction of generating cis-5-hydroxy-L-pipecolic acid by using L-pipecolic acid as a substrate.
A method for producing cis-5-hydroxy-L-pipecolic acid or a pharmacologically acceptable salt thereof, or a solvate thereof, which comprises the step of allowing the protein according to claim 11 to act on L-pipecolic acid to generate cis-5-hydroxy-L-pipecolic acid.
The production method according to claim 12, which further comprises:
the step of allowing a protein having an activity of catalyzing a reaction of generating L-aminoadipate-delta-semialdehyde by using L-lysine as a substrate to act on L-lysine to generate L-aminoadipate-delta-semialdehyde, and then converting L-aminoadipate-delta-semialdehyde into delta-1-piperideine-6-carboxylic acid; and

the step of allowing a protein having an activity of catalyzing a reaction of generating L-pipecolic acid by using delta-1-piperideine-6-carboxylic acid as a substrate to act on the obtained delta-1-piperideine-6-carboxylic acid to generate L-pipecolic acid.
The gene recombinant microorganism or gene recombinant Escherichia coli according to claim 9 or 10, which has an ability to produce 50 mg or more of cis-5-hydroxy-L-pipecolic acid per 1 L of culture medium.